Malachite green mediates homodimerization of antibody V_L domains to form a fluorescent ternary complex with singular symmetric interfaces

We report that a symmetric small-molecule ligand mediates the assembly of antibody light chain variable domains (V_Ls) into a correspondent symmetric ternary complex with novel interfaces. The L5* fluorogen activating protein is a V_L domain that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two V _L domains. Unliganded L5* V_L domains crystallize as a similar antiparallel V_L/V_L homodimer. The complementarity-determining regions are spatially oriented to form novel V _L/V_L and V_L/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable V_L/MG/V_L complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two V_L domains into a single protein tightens MG binding over 1000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high-affinity protein tags and capture reagents.